There are several known techniques to assay viruses, bacterial cells and spores in environmental samples. The techniques may also be applied to biological fluids. Overall, they may be divided into three main categories.
The first category involves direct visualization of pathogens by direct optical, electron microscopy or atomic force microscopy. The second category involves detecting specific genes or oligonucleotide sequences after polymerase chain reaction (PCR) amplification. The third category involves common methods in assay of pathogens based on the use of pathogen-specific antibodies. This third type may employ various techniques, such as radioimmunoassay (RIA), enzyme-linked immunosorbant assay (ELISA), immunofluorescent microscopy, etc.
Sensitivity of all known detection methods may depend on the efficiency of pathogen collection, as well as the level of sensitivity of the detection method. Generally, as the level of sensitivity demanded increases, the sample volume decreases. In essence, when the pathogen concentration is low, deposition of pathogens may become more difficult.
Take, for instance, electron microscopy, where the total microscope grid size (S) can be approximately 3-5 mm2. While such grid can float over a large sample volume, the surface density of particles in T seconds can be denoted byN/S˜C(DT)1/2  (1)
where N represents the total number of bound particles, S represents the total open (viewable) area of a microscope grid, C represents the pathogen concentration, D represents the diffusion coefficient of the particles in solution, and T represents time.
D can be low for even for relatively small pathogens, such as viruses. For example, D can be 10−12 m2/sec for virus particles. In this case, it can take a long time to accumulate sufficient density of bound viruses. Thus, to have at least one pathogen per square micron captured in, for example, 30 min., C may need to exceed 2×1010 particles/mL. This pathogen concentration may be needed to overcome diffusion limitation, no matter how large the sample volume is. If the sample volume is 1 mL, then approximately 5×106 particles out of 2×1010 particles may be captured under these conditions.
Atomic force microscopy (AFM) can present another challenge to sensitivity increase. Although it has the ability to detect single viruses, one would probably need to have at least 106 viruses/mL to be able to image a few viral particles in a 5×5 μm2 image suitable for observation. In contrast to electron microscopy, relatively slow scanning in AFM does not tend to allow one to quickly search a large area.
Single particle sensitivity has also been introduced using other techniques. For example, conductivity of a gap between two nanowires was shown to be sensitive to the binding of a single viral particle. However, similar to the above microscopy techniques, this technique usually works only with highly concentrated solutions when particles could appear on a small stage between the nanowires in a reasonably short time.
In essence, a major limitation with all known detection techniques that are sensitive to a single pathogen is that single pathogens are hard to bring to view when pathogens are spread within highly diluted solutions or suspensions.
An approach to overcome this limitation is preconcentrating the samples. This procedure is common in environmental analyses. However, additional preconcentration prolongates analysis and tends to be costly.